Our long-term goal is to understand the cellular mechanisms involved in the pathogenesis of pulmonary hypertension. Previous studies have characterized morphologic and functional changes that occur in the pulmonary vasculature during the development of idiopathic pulmonary arterial hypertension (IPAH), but the cellular mechanisms underlying these changes remain poorly understood. Using animal models, we recently demonstrated a role for alterations in pulmonary arterial endothelial (EC) and smooth muscle cell (SMC) intracellular pH (pHi) homeostasis, due to increased Na+/H+ exchanger isoform 1 (NHE1) expression. In addition, we have also shown that these cells exhibit elevated intracellular Ca2+ concentration and increased expression of calpains, a family of cytosolic, Ca2+-activated, neutral cysteine proteases. Changes in pHi, Ca2+ homeostasis and calpain activity have profound influences on cell function, although whether these pathways are altered during IPAH and the mechanism by which they modulate cell growth and migration are poorly understood. In animal models of pulmonary hypertension, we found that inhibition of calpain reduced right ventricular hypertrophy and pulmonary vascular remodeling. Recently, studies have suggested that NHE1 could act as a membrane anchor independent of its ion translocation properties. NHE1 co-localizes with and binds to ezrin, a protein which contains multiple binding domains and is important mediator of protein-protein interactions. Of particular interest, the C-terminal of ezrin contains a binding site for filamentous actin, providing a possible link between NHE1 and cytoskeletal rearrangement. If true, increased NHE1 and/or ezrin expression/interactions during IPAH could create conditions favorable for membrane/cytoskeleton interactions. Moreover, calpain activation is influenced by pHi, and results in Rho kinase activation, which in turn facilitates ezrin activation and binding with targe proteins. Thus, we hypothesize that during IPAH, upregulation of NHE1 and calpain, and interactions between these pathways, lead to enhanced EC and SMC migration and proliferation, hallmarks of disease development and progression. To test this hypothesis, we will use a combination of techniques including confocal microscopy, FRET, knockdown and overexpression approaches, co-immunoprecipitation and migration and proliferation assays in pulmonary vascular ECs and SMCs and lung tissue and mRNA from IPAH and control subjects to determine: 1) whether calpain activity is enhanced in EC and SMCs from patients with IPAH and whether inhibition through genetic or pharmacologic means alters proliferation and migration in these cells; 2) whether NHE1 expression, activity and interactions with ezrin and actin are altered in vascular cells from IPAH patients and, if so, the involvement of these changes in cell function and 3) whether there is an interaction or linkage between the Ca2+/calpain and NHE1/ezrin pathways that contributes to the functional changes observed in ECs and SMCs from IPAH patients. PUBLIC HEALTH RELEVANCE: The experiments in this proposal will explore cellular mechanisms involved in the development of idiopathic pulmonary arterial hypertension (IPAH), a devastating disease with limited treatment options. The availability of tissues and cells from IPAH patients promises to reveal novel information that will further our understanding the cellular changes that occur in the pulmonary vasculature with development of IPAH. Such information is crucial to advancing treatment and developing new therapeutic options to treat this deadly disease. (End of Abstract)